Ž .Brain Research 822 1999 80–87

Research report

Localization of a-tocopherol transfer protein in the brains of patients withataxia with vitamin E deficiency and other oxidative stress related

neurodegenerative disorders

Richard P. Copp a, Thomas Wisniewski b, Faycal Hentati c, Abdelmajid Larnaout c,Mongi Ben Hamida c, Herbert J. Kayden a,)

a Department of Medicine, New York UniÕersity Medical Center, 550 First AÕenue, New York, NY 10016, USAb Departments of Neurology and Pathology, New York UniÕersity Medical Center, New York, NY 10016, USA

c Laboratoire de Neuropathologie et Biologie Moleculaire, Institut National de Neurologie-La Rabta, 1007, Tunis, Tunisia

Accepted 22 December 1998

Abstract

Ž .Vitamin E a-tocopherol is an essential nutrient and an important antioxidant. Its plasma levels are dependent upon oral intake,Ž .absorption and transfer of the vitamin to a circulating lipoprotein. The latter step is controlled by a-tocopherol transfer protein a-TTP ,

which is a 278 amino acid protein encoded on chromosome 8, known to be synthesized in the liver. Mutations in a-TTP are associatedŽ .with a neurological syndrome of spinocerebellar ataxia, called ataxia with vitamin E deficiency AVED . Earlier studies suggested that

a-TTP is found only in the liver. In order to establish whether a-TTP is expressed in the human brain, and what relationship this has toAVED, we studied immunohistochemically the presence of a-TTP in the brains of a patient with AVED, normal subjects, and patients

Ž . Ž . Ž . Ž .with Alzheimer’s disease AD , Down’s syndrome DS , cholestatic liver disease CLD and abetalipoproteinemia ABL . Theneuropathology of both AD and DS is thought to be related in part to oxidative stress. The diseases of AVED, of cholestatic liver disease,and of abetalipoproteinemia are thought to be due to lack of circulating tocopherol, leading to inadequate protection against oxidativedamage. We demonstrate the presence of a-TTP in cerebellar Purkinje cells in patients having vitamin E deficiency states or diseasesassociated with oxidative stress. q 1999 Elsevier Science B.V. All rights reserved.

Keywords: Tocopherol transfer protein; Ataxia with vitamin E deficiency; Familial isolated vitamin E deficiency; Vitamin E; Alzheimer’s disease; Down’ssyndrome; Cholestatic liver disease; Abetalipoproteinemia

1. Introduction

The study of neurodegenerative diseases associated withincreased oxidative stress has drawn many investigators.The present report addresses the potential role of a-

Ž .tocopherol Vitamin E in the human brain, by establishingŽ .the presence of the a-tocopherol transfer protein a-TTP

Ž .in diseased human brains. Plasma vitamin E a-tocopherollevels are regulated by its oral intake, absorption and

w xtransfer into a circulating lipoprotein 20 . The last step iscontrolled by a-TTP, whose 278 amino acid composition,genetic localization at chromosome site 8q13 and DNA

w xsequence have been identified 1,13,31 . Mutations in thegene for a-TTP result in a neurologic syndrome of spino-

) Corresponding author. Fax: q1-212-263-6751

w xcerebellar ataxia 13,16,31,39,47 , called ataxia—with vi-Ž .tamin E deficiency AVED . This condition is character-

ized by progressive ataxia, areflexia, dysarthria, sensoryw xloss and pyramidal signs 8,31,44 . The symptoms of

AVED are similar to those of Friedreich’s ataxia; however,these two conditions can be distinguished by analysis ofthe frataxin gene on chromosome 9 where large GAArepeats are found in the first intron of Friedreich’s ataxia

w xpatients 21 and by a measurement of the plasma a-w xtocopherol levels 8 . These a-tocopherol levels are usually

w xbelow one mgrml in AVED patients 8,15 , while normalw xlevels are 4–9 mgrml 10 . The largest group of AVED

patients is found in North Africa and share a commongenetic mutation for a-TTP, the 744delA mutation, whichleads to a substitution with 14 incorrect amino acids aftercodon number 248 in the protein. However, different

0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved.Ž .PII: S0006-8993 99 01090-2

( )R.P. Copp et al.rBrain Research 822 1999 80–87 81

mutations in a-TTP have been found among various ethnicwgroups in Europe, North America and Asia 8,13,16,

x31,39,47 .a-Tocopherol is well established as the major lipid–

soluble chain reaction-breaking antioxidant, and it is im-portant for protecting the integrity of lipid structures,

w xespecially membranes 4 . The neurological consequencesof its deficiency were originally characterized in patients

Ž . w xwith the genetic disorder, abetalipoproteinemia ABL 3Ž . w xand in cholestatic liver disease CLD 35 . The lack of

vitamin E results initially in a peripheral neuropathy due tothe dying back of large calibre axons with subsequent loss

w xof central neurons 19,30 . We have previously measuredthe concentrations of a-tocopherol in biopsies of the suralnerve in patients with AVED and homozygous hypobeta-lipoproteinemia, and found them to be reduced even before

w xaxonal degeneration has occurred 44 . How a-tocopherolenters the nervous system is not known, though a-tocopherol delivery to certain cells is carried out by

w x w xlipoprotein lipase 42 and the LDL receptor 41 , and theLDL receptor has been reported to deliver lipids across the

w xblood–brain barrier 9 .Earlier studies suggested that a-TTP was found exclu-

w xsively in the liver cytosol 7,29 , and that it functioned tow xtransfer a-tocopherol into nascent VLDL 43 . Hence, the

neurological symptoms of AVED were thought to berelated to a lack of vitamin E delivery to the centralnervous system. However, studies of extracts of rat andrabbit organs including lung, heart and brain showed thatbinding activities were present for radiolabelled a-

w xtocopherol in each of these organs 11,14,29,48 . We there-fore looked whether a-TTP is expressed in the humanbrain using a specific rabbit polyclonal antibody to recom-binant human a-TTP. Using this antibody, we immunohis-tochemically demonstrate the presence of a-TTP in thehuman brain of a patient with the 744delA mutation whodied of AVED, and in patients with Alzheimer’s diseaseand Down’s syndrome. Each of these conditions is associ-ated with oxidative injury of the brain. We also demon-strate the presence of a-TTP in two diseases associatedwith low vitamin E levels, cholestatic liver disease andabetalipoproteinemia. In contrast, among age-matched con-trol patients, we did not find a-TTP in the brain, usingimmunohistochemical techniques.

2. Materials and methods

2.1. Expression and purification of recombinant humana-TTP

The cDNA coding for human a-TTP in vector pSPORT1 was digested with SphI and Sal I, and recloned into themultiple cloning site of the expression vector pPROEX-1

Žcontaining a 6= histidine tag coding sequence Life Tech-

.nologies, Gaithersburg, MD, Genbank Accession U19585 .The resultant construct was redigested with Sal I, bluntended with the Klenow enzyme, and religated to producea-TTP in the proper reading frame. This construct wastransformed into E. coli DH5a by calcium phosphateprecipitation, and a clone confirmed by DNA sequencing.Protein expression was induced with 1 mm IPTG for 3 h.Recombinant a-TTP was extracted by a combination offreezerthaw and sonication and purified by Ni–NTA nickel

Ž .affinity resin Quiagen, Chatsworth, CA followed by SDSw xpolyacrylamide gel electrophoresis 23 .

2.2. Preparation of antibodies

Ni–NTA nickel affinity resin- and SDS gel-purifiedrecombinant human a-TTP was used to induce polyclonalantibodies in New Zealand white female rabbits usingFreund’s complete adjuvant. The preparation of mono-

w xclonal antibody AT-R1 against rat a-TTP 36 , whichw xcross-reacts with the human protein 1 has been described

previously.

2.3. Preparation of deletion mutants of recombinant hu-man a-TTP

Human a-TTP in vector pSPORT 1 was digested withrestriction enzymes SphI and Sal I, and recloned into theSal IrSphI sites of the double-stranded form of the bacte-riophage M13MP19. A single-stranded preparation of aclone was subjected to phosphorothioate in vitro mutagen-

w x Ž .esis 37 using a commercial kit Amersham Life Scienceand a 33 base oligonucleotide corresponding to humana-TTP cDNA bases 727–760, with base 744 deleted. Adouble-stranded form of the mutated clone in M13MP19was confirmed by sequencing and then digested withSal IrSphI. The gel-purified insert was cloned into expres-sion vector pPROEX-1 in the SalI and the SphI locationof the multiple cloning site. After transformation into E.coli DH5a , the resultant construct was redigested withSal I, blunt ended with the Klenow enzyme, and religatedto produce a-TTPdel744A in the proper reading frame,which results in 14 incorrect amino acids after residue 248followed by an ochre stop codon. This construct wastransformed into E. coli DH5a and confirmed by sequenc-ing. The same procedure was used to produce the deletionmutant corresponding to the mutation, a-TTP530 AG)6,in the expression vector pPROEX-1, in which two incor-rect amino acids are added after residue 176, followed byan ochre stop codon.

2.4. Preparation of cell extracts and Western blotting

HeLa and HepG2 cells originated from American TypeCulture Collection and were cultured in Eagle’s minimal

( )R.P. Copp et al.rBrain Research 822 1999 80–8782

w xessential medium containing 10% fetal calf serum 33 .Tissue culture cells were homogenized in 0.25 M sucroseusing a motor-driven teflon-glass homogenizer and frac-

w xtionated using differential centrifugation by velocity 5 .w xProtein was determined by the method of Lowry et al. 26

and proteins of fractionated extracts further separated byw xSDS polyacrylamide gel electrophoresis 23 and trans-

w xferred to PVDF membranes 34,40 . Western blotting andanalysis of cell extracts on PVDF membranes used a 1:400dilution of the AT-R1 mouse monoclonal anti-rat-a-TTPand goat anti-rabbit-horseradish peroxidase-chemilumines-

w xcence labeling 45 . AT-R1 and goat anti-mouse-alkalinephosphatase were also used in a similar manner with 4-nitroblue tetrazoliumr5-bromo-4-chloro-3-indolyl phos-

Ž . w xphate NBTrBCIP staining 2,34 . Direct extraction ofIPTG-induced E. coli containing a-TTP expression vec-

Ž . Ž .tors 1 mM, 3 h was with 1:1 VrW 2-fold SDS gelw xsample buffer 23 . Controls for the Western blotting in-

cluded absorption of the monoclonal antibody AT-R1 withpurified recombinant a-TTP.

2.5. Patient tissue samples

Human liver was obtained from the Bellevue Hospital,New York, NY, transplant registry. Normal human brainsections and sections from patients with AD and DS wereobtained from the Department of Neuropathology at NewYork University Medical Center, New York, NY and theInstitute for Basic Research in Developmental DisabilitiesŽ .Staten Is., NY . Autopsy sections of cerebellum, temporalcortex, hippocampus, brainstem and spinal cord of a 29-year-old patient with the complete neurological syndrome

Ž .of AVED and with a mutation in the a-TTP gene del744Awere provided by Dr. Abdelmajid Laranout and previously

w x Ždescribed 24 . Brain tissue from five AD subjects three

.males and two females; ages 75, 72, 69, 79 and 85 werestudied—and similar findings were present in each patient.

w xAll AD patients fulfilled the CERAD criteria of AD 28 .ŽThe five DS patients ages 61, 62, 64, 64, and 69; cases

. w xno. 36–40 have been previously described 46 . The brainsections from an eight-year-old male who died ofcholestatic liver disease and had the characteristic neuro-

w xlogical findings 35 were obtained from Dr. James Nelsonof Louisiana State University Medical Center. Brain sam-ples of the patient with abetalipoproteinemia, a 43-year-oldwoman who had the neurologic syndrome of spinocerebel-

w xlar ataxia as a consequence of vitamin E deficiency 3 ,were also provided by Dr. James Nelson. Brain sections of

Žfive neurologically normal controls three males and two.females; ages 65, 72, 80, 78, and 79 were also studied.

2.6. Immunocytochemistry

Immunocytochemistry was performed using a 1:400dilution of rabbit polyclonal anti-a-TTP as primary anti-body and a 1:200 dilution of biotinylated goat anti-rabbitŽ .Vector Laboratories, Burlingame, CA as secondary anti-body. Paraffin-embedded sections were cleared in xylene,hydrated through graded ethanol baths, and washed threetimes for 10 min in Tris-buffered saline pH 7.4–0.05%

Ž .Tween 20 TBST before analysis. Blocking was for 16 hŽ .at 48C using 3% bovine serum albumin BSA in TBST.

Following three 10-min washes in TBST, primary antibodybinding was for 16 h at 48C in 2% goat serum–0.1% BSAin TBST. After three 10-min washes in TBST, secondaryantibody binding was for 1 h in 5% fetal calf serum–5%goat serum–0.1% BSA in TBST. Following three 10-min

w xwashes in TBST, immunoperoxidase ABC staining 17,18using diaminobenzidine was carried out according to the

Ž .manufacturer’s directions Vector Laboratories . Some sec-

Fig. 1. Western blots of cytosol extracts. Proteins from cytosolic extracts of tissues and tissue culture cells, direct SDS extracts of E. coli containingrecombinant a-TTP expression vectors and Ni-NTA affinity purified recombinant a-TTP were separated by SDS gel electrophoresis, Western-blotted, and

Ž . Ž .probed with AT-R1 mouse monoclonal anti-rat-a-TTP and either chemiluminescence Panel A or colorimetric detection Panel B . Panel A-lane 1, 75 mgcytosolic extract of HepG2 cells; lane 2, 0.03 mg affinity purified recombinant human a-TTP; lane 3, 3 mg of rat liver cytosol; lane 4, 100 mg of humanfetal liver cytosol; lane 5, molecular weight standards. Panel B-SDS extracts of E. coli expressing recombinant human a-TTP preparations. Lane 1,full-length a-TTP; lane 2, a-TTPdel744A; lane 3, a-TTP530AG)6; lane 4, molecular weight standards.

( )R.P. Copp et al.rBrain Research 822 1999 80–87 83

tions were counterstained for 30 s with 10% Harris hema-toxylin. Controls for the immunocytochemistry includeduse of a 1:400 dilution of preimmune serum and absorp-

tion of the rabbit anti-a-TTP with the purified recombinanta-TTP. Sections were also subjected to hematoxylin–eosin

w xstaining and to modified Bielschowsky staining 46 .

Fig. 2. Immunocytochemistry of AVED and Alzheimer disease patients’ brain sections. Formalin-fixed, paraffin embedded sections were analyzed forŽ .a-TTP immunoreactivity using rabbit polyclonal anti-a-TTP at 1:400 dilution. Panel A, B, cerebellar sections from AVED patient. A Probed with

anti-a-TTP. The arrow points to granular cytoplasmic immunoreactivity seen in Purkinje cell cytoplasm. An insert in the upper left corner shows theŽ .Purkinje cell at higher magnification. B A sequential section to A probed with anti-a-TTP serum pre-absorbed with recombinant a-TTP. No specific

Ž .immunostaining is noted. The sections are counterstained with hematoxylin. Scale bars: 50 mM. Panel C, D, cerebellar sections from an AD patient Cprobed with anti-a-TTP. The arrows point to immunostaining of Purkinje cells. An insert in the upper left corner shows the Purkinje cell at higher

Ž .magnification. D A sequential section to C probed with anti-a-TTP absorbed with recombinant a-TTP. No specific immunostaining is noted. Thesections are counterstained with hematoxylin. Scale bars: 50 mM. Panel E, F, hippocampal sections from the Alzheimer’s disease patient shown in panels

Ž . Ž .C, D. E Probed with anti-a-TTP. The arrows points to diffuse immunoreactivity seen in CA2 neurons. F A sequential section to E probed withpreimmune serum. Scale bars: 50 mM. Panel G, cerebellar section from a 72-year-old control patient. The arrow points to Purkinje cells which show nospecific immunoreactivity. The section is counterstained with hematoxylin. Scale bar: 50 mM.

( )R.P. Copp et al.rBrain Research 822 1999 80–8784

3. Results

( )3.1. Western blot analysis of cytosol extracts Fig. 1

Cytosol extracts of tissue culture cells or standards wereprepared and proteins separated by a 10% SDS polyacryl-amide gel and transferred to PVDF membranes using a1:400 dilution of anti-rat a-TTP mAb, AT-R1, and chemi-luminescent detection. In addition, rabbit polyclonal anti-human a-TTP antibody was used to analyze Western blotsof extracted proteins. Its use permitted ready detection of amolecular weight 32,000 immunoreactive protein from ratliver cytosol extracts; while the antibody could not detectan a-TTP-immunoreactive protein from the same quanti-ties of rat liver nuclear, mitochondrial, or microsomal

Ž .fraction proteins data not shown . The cross-reaction ofthe rabbit polyclonal anti-human a-TTP to rat liver a-TTPis not unexpected because human and rat a-TTP have a94% amino acid sequence similarity and antibodies to rata-TTP cross-react to the human a-TTP; the monoclonalantibody to the rat a-TTP, AT-R1, cross-reacts to the

w xhuman a-TTP 1 , and a polyclonal antibody to a sepa-rately isolated rat a-TTP isoform cross-reacts to indepen-

w xdently isolated human liver a-TTP 22 .As shown in Fig. 1A,B, a Western blot of cytosolic

Ž .extract of HepG2 human hepatoblastoma lane A1 , and ratŽ .liver lane A3 , exhibit a specific immunoreactive band

with a molecular weight of 32,000, corresponding to a-TTP. Ni–NTA nickel affinity purified recombinant human

Ž .a-TTP lane A2 exhibited a band corresponding to amolecular weight of 35,000. The molecular weight of the

Fig. 3. Immunocytochemistry of cerebellar sections from DS, CLD, and ABL patients. Panel A, Shows a cerebellar section from a patient with DS probedwith anti-a-TTP. The arrow points to a Purkinje cell which has diffuse cytoplasmic immunoreactivity. The section is counterstained with hematoxylin.Scale bars: 50 mM. Panel B, cerebellar section from a patient with CLD probed with anti-a-TTP and counterstained with hematoxylin. The arrows point tothe cell body and dendrite of a Purkinje cell. Scale bar: 50 mM. Panel C, cerebellar section from a patient with ABL probed with anti-a-TTP. The arrowspoint to the immunoreactive Purkinje cells. Scale bars: 50 mM.

( )R.P. Copp et al.rBrain Research 822 1999 80–87 85

recombinant human a-TTP protein is 35,363, instead of31,749 for the native protein, because of additional aminoacids in the construct containing the 6= histidine tag.Human fetal liver exhibited a major immunoreactive bandat 30,000 and a band of much less intensity at approxi-mately 18,000 molecular weight; their locations are pre-

Ž .sumably due to a-TTP degradation lane A4 . The twomost prevalent mutations in the a-TTP gene among pa-tients with AVED are the a-TPPdel744A and the a-TPP530AG)6. We have generated these two mutationsand expressed them in E. coli. Fig. 1B shows the Western

Ž .blot of normal lane B1 and of these two mutations inlanes B2 and B3, respectively. Lane 4 has the molecularweight markers.

3.2. Immunocytochemistry

Immunocytochemical analysis of human brain cerebel-Ž .lar cortical sections in the patient with AVED Fig. 2A

Ž . Ž .and in both the AD Fig. 2C and DS Fig. 3A patientsdemonstrate specific staining of a-TTP in Purkinje cells.The immunoreactivity of the Purkinje cells in the AVEDpatient was distinct and different from that found with theAD and DS patients. In the AVED patient, the cytoplasmicstaining of the Purkinje cells was more granular andpunctate, suggesting a possible cytoplasmic organelle lo-

Ž .calization Fig. 2A while in the AD and DS patient cells,Ž .the staining was much more diffuse Fig. 2C, Fig. 3A .

The immunoreactivity of the Purkinje cells was moreprominent in the DS patients compared to the AD patients,correlating with the much more numerous amyloid de-posits in the cerebellum, among the DS patients. Somepyramidal neurons in the hippocampus of the AD and DSpatients also showed faint diffuse cytoplasmic stainingŽ .Fig. 2E in an AD patient . The temporal cortex, hip-pocampus and brainstem sections of the AVED patient didnot show any positive immunohistochemical reaction withthe anti-a-TTP antibody, as would be expected since theseareas of the brain are not affected in AVED patients.

Ž .Cerebellar cortical sections from both CLD Fig. 3CŽ .and ABL Fig. 3D patients demonstrate diffuse cytoplas-

mic staining for a-TTP in Purkinje cells. The immuno-Ž .staining in the CLD patient Fig. 3C demonstrates that the

a-TTP immunoreactivity extends throughout the PurkinjeŽ .cell dendrites. As shown in the ABL patient Fig. 3D , not

all Purkinje cells are immunoreactive for a-TTP, andreactive cells may appear in clusters. The a-TPP immuno-reactivity was observed in only about 10% of Purkinjecells among the CLD and ABL patients.

The above immunohistochemical staining was specific,as it was not seen when preimmune serum was used and itcould be specifically absorbed with the recombinant a-TTPŽ .see Fig. 2B,D,F . In normal brain tissue no staining wasevident, suggesting that the level of a-TTP was below thelevel of detection.

4. Discussion

The symptoms of AVED are dominated by centralnervous system dysfunction. This disorder is directly linkedto low vitamin E levels resulting from mutations in a-TTP.a-TTP is known to be synthesized in the liver and it isthought that the mutated a-TTP in the liver of AVEDpatients caused a failure of a-tocopherol to be incorpo-rated into VLDL particles, resulting in sharply lowerplasma vitamin E levels, leading to a reduced delivery ofvitamin E to the CNS. This presumably renders the neu-ronal cells more susceptible to oxidative injury, because oftheir higher energy use and lipid content. In this paper, wedemonstrate that, although a-TTP could not be detected innormal brains, it was clearly present in the brain of apatient with AVED, in its mutated form and in the brains

Žof patients with marked vitamin E deficiency CLD and.ABL , as well as in patients with neurodegenerative dis-

Žeases thought to be associated with oxidative stress AD.and DS . The a-TTP was found to be expressed in Purk-

inje cells as well as in pyramidal cells of the hippocampus.This demonstration of protein expression suggests thata-TTP may be produced locally in the brain and have arole in the binding and transport of a-tocopherol both intraand intercellularly.

Prior studies using polyclonal and monoclonal antibod-ies to a-TTP failed to detect this protein in extracts of

w xtissues other than liver, including whole brain extracts 1 .However, these negative results are related to the lowerlevels of a-TTP in the brain and the fact that a-TTP isconcentrated in only a small number of cell types. It wasonly with fractionation of hepatic cells and examination ofthe cytosolic extract that the a-TTP was sufficiently con-centrated to allow detection. Prior Northern blot studiesalso failed to detect a-TTP in extracts of tissues other than

w xliver, including rat brain 48 . These negative results arealso likely to be due to the low levels of a-TTP in thebrain and the limited number of cells expressing it.

In normal brain tissue, we failed to detect any immuno-histochemical staining with the anti-a-TTP antibodies. Thelack of a-TTP immunoreactivity in normal brain mostlikely represents a low level of expression and a lack ofsensitivity of our methods. a-TTP could be detected im-munohistochemically only in a limited number of braincell types in neurodegenerative diseases associated withoxidative stress where a-TTP expression is presumablyincreased. The immunoreactivity we identify with ourantibody is specific since it could be fully absorbed by thepurified protein and was not found using preimmune serum.

The Purkinje cells are an important site of pathology inthe AVED patient, with extensive Purkinje cell loss being

w xreported at autopsy 24 , and which we corroborated in ourneuropathological evaluation of this patient’s cerebellum.This correlates clinically with cerebellar ataxia being a

w xmajor symptom of AVED 8 . In AVED, the mutated and

( )R.P. Copp et al.rBrain Research 822 1999 80–8786

poorly functioning a-TTP could be up-regulated in re-sponse to oxidative stress associated with a vitamin Edeficient state. The mutated and conformationally altereda-TTP appears to accumulate within cytoplasmic or-

Ž .ganelles see Fig. 2A .The granular immunoreactivity in the Purkinje cells of

the AVED patient is distinct from the more diffuse stain-ing of the Purkinje cells in the AD and DS patients. ThePurkinje cell a-TPP immunoreactivity seen among the ADand DS patients is likely to be related to the oxidativestress these cells are thought to undergo. Despite theirnormal appearance, the Purkinje cells are known to be asite of damage in AD and DS. About 5–10% of Purkinjecells from AD and DS patients contain abnormally in-creased numbers of hydrolase-positive lysosomes due to

w xprogressive metabolic compromise 6 . Significantly theobserved a-TTP immunoreactivity was more prominent inthe DS patients where the cerebellar amyloid burden was

Ž .much higher. In both DS and in AD Fig. 2E patients,staining was also seen among some hippocampal neurons.This correlates with the hippocampus being the site ofmost prominent oxidative damage in these conditionsw x12,25,32,38 . The CLD and ABL patients both representdisease states resulting in low plasma a-tocopherol levelsand both showed diffuse cytoplasmic a-TTP immuno-reactivity of Purkinje cells. The latter immunohistochemi-cal findings, and the results of the AD and DS patients,suggests that diseases associated with oxidative stress re-sult in increased cytoplasmic a-TTP in certain cell popula-tions.

Recent studies of Friedreich’s ataxia have shown thatthere is decreased expression of frataxin in the mitochon-

w xdria leading to increased iron levels 21 . This increase iniron could result in increased oxidative stress and freeradical toxicity to neurons, resulting in the neurologicsyndrome of ataxia. The mutations in the a-TTP in AVEDpatients may equally predispose the same neurons to freeradical damage because of an inability to utilize the sharplydiminished content of a-tocopherol being delivered to thecentral nervous system. The striking similarity in the neu-rologic syndromes between these two genetic disorders hascaused confusion in establishing the correct diagnosis. It isimportant to establish that vitamin E deficiency is thecause of the neurologic abnormalities, since supplementa-tion with vitamin E halts progression of AVED, results inimprovement in the symptomology, and prevents the de-velopment of neurologic signs in those at risk when treat-ment is initiated in childhood before damage occursw x8,15,16,27 .

Our studies demonstrate for the first time the presenceof a-TTP within cells of the CNS. These immunohisto-chemical findings suggest that a-TTP can be synthesizedin the brain, as well as in the liver; albeit at much lowerlevels. The demonstration of a-TTP production within theCNS provides insight into why the central nervous systemis such a major site of dysfunction in conditions where the

a-TTP is mutated. In addition, our findings expand therole of a-TTP beyond the incorporation of vitamin E intoVLDL particles in the plasma, and suggest that it also hasa local role in the response of the CNS to oxidative stress.

Acknowledgements

This work has been supported by Muscular DystrophyŽ .Association to F.H. , the Tunisian State Secretary of

Ž .Research, and NIH Grant AG15408 T.W. . We thank Dr.Ž .Hiroyuki Arai University of Tokyo, Tokyo, Japan for

donation of recombinant human a-TTP in vector pSPORT1 and monoclonal antibody AT-R1 and Dr. James Nelsonof Louisiana State University Medical Center for providingthe brain samples from the CLD and ABL patients.

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